key: cord-0800061-7clr6hbf
authors: Cutolo, Edoardo Andrea; Mandalà, Giulia; Dall’Osto, Luca; Bassi, Roberto
title: Harnessing the Algal Chloroplast for Heterologous Protein Production
date: 2022-03-30
journal: Microorganisms
DOI: 10.3390/microorganisms10040743
sha: 3ee7a6e3d59c54a9ea39b1b904f357519cea0249
doc_id: 800061
cord_uid: 7clr6hbf

Photosynthetic microbes are gaining increasing attention as heterologous hosts for the light-driven, low-cost production of high-value recombinant proteins. Recent advances in the manipulation of unicellular algal genomes offer the opportunity to establish engineered strains as safe and viable alternatives to conventional heterotrophic expression systems, including for their use in the feed, food, and biopharmaceutical industries. Due to the relatively small size of their genomes, algal chloroplasts are excellent targets for synthetic biology approaches, and are convenient subcellular sites for the compartmentalized accumulation and storage of products. Different classes of recombinant proteins, including enzymes and peptides with therapeutical applications, have been successfully expressed in the plastid of the model organism Chlamydomonas reinhardtii, and of a few other species, highlighting the emerging potential of transplastomic algal biotechnology. In this review, we provide a unified view on the state-of-the-art tools that are available to introduce protein-encoding transgenes in microalgal plastids, and discuss the main (bio)technological bottlenecks that still need to be addressed to develop robust and sustainable green cell biofactories.

The advent of recombinant DNA technology in the 1970s revolutionized biological research and paved the way for modern biotechnology. Starting from the pioneering assembly of artificial circular DNA molecules in vitro [1] and their autonomous replication in a living host [2] , genetic engineering achieved its first landmark with the heterologous synthesis of the human peptide hormone somatostatin in a bacterium [3] . The ability to introduce foreign genes in simple biological hosts-typically fast-growing, easily cultivable microorganisms-is paramount to overcome the inherent low yields of the naturally producing species, and to allow the upscaling of production systems.

The last decade has witnessed significant advancements in the genetic engineering of photosynthetic microbes, notably of eukaryotic microalgae of the phylum Chlorophyta, to serve as alternative heterologous hosts [4] to the commonly employed bacteria, yeast, and mammalian cell lines. Several microalgal species are GRAS (generally regarded as safe) organisms and, being oxygenic phototrophs, lead a frugal lifestyle based on light-powered fixation of atmospheric CO 2 , with minimal input requirements compared to heterotrophs. Furthermore, when moved from their natural habitats to controlled (non-nutrient limited) conditions, microalgae display increased growth rates, being suitable for mass culture. Various algal species are amenable to genetic engineering [5] , and new strains can be created in a matter of weeks and readily tested for the expression of the products of interest.

Most Chlorophyta contain a large chloroplast-the site of photosynthetic reactions and other metabolic processes-which harbors a compact, tractable genome known as the plastome. Three features render this organelle a preferential target for the expression of recombinant proteins compared to the nucleus: (i) its polyploid condition, (ii) the recombinationmediated targeting of foreign DNA sequences, (iii) the absence of silencing effects.

A prerequisite to develop efficient chloroplast expression strategies is the understanding of the features and workings of this peculiar genetic system. Chloroplast engineering owes much to basic research conducted on plants and on the eukaryotic microalga Chlamydomonas reinhardtii [6, 7] ; today, this species serves as an experimental model with a rich and dedicated genetic engineering toolbox. In this review, we provide a transdisciplinary update on the available strategies to express recombinant products in microalgal plastids (summarized in Figure 1 ), highlighting the recent advancements in multigenic engineering, inducible expression systems, and biotechnological transfer to non-model strains. We also discuss areas that require major technological innovation and introduce long-term goals that will contribute to developing more robust algal cell factories.

Known as semi-autonomous organelles, algal chloroplasts are the remnants of a photosynthetic cyanobacteria engulfed by a heterotrophic protist [8] . The bacterial ancestry of plastomes is reflected by the strong AT-rich codon usage bias; therefore, codon optimization is an essential step in the design of chloroplast transgenes [9] . Striking prokaryotic traits of plastids include the transcription of operons in the form of polycistronic units [10] , and the bacterial-like composition of the ribosomes [11] causing analogous susceptibility to antibiotics [12, 13] . The coevolutionary process between the endosymbiont and the eukaryotic host determined a major horizontal gene transfer to the nucleus [14] , resulting in the shrinkage of plastomes to a core set of approximately 80-100 genes. These mostly encode subunits of the photosynthetic apparatus and components of the chloroplast transcriptiontranslation machinery, including the single, plastid-encoded RNA polymerase (PEP) [15] . Nuclear gene expression is thus responsible for the synthesis of most plastid proteins (≈3000) [16] , which are imported into the organelle as precursors [17, 18] .

In C. reinhardtii, plastid genes are arranged on a circular chromosome of 204 kb [19] maintained at high polyploidy (≈83 copies) [20] , which is uniparentally inherited in sexually reproducing microalgae [21] . Algal plastomes typically display a quadripartite structure, with small and large "single copy" regions divided by two large "inverted repeat" (IR) regions that bear identical gene complements, although the overall genomic architecture can vary significantly between species [22, 23] . In C. reinhardtii, almost 20% of the plastome consists of non-coding, short dispersed DNA repeats [19] . In addition, to perform structural roles in mRNAs [24] , these elements assist in the maintenance of genome integrity by facilitating the conservative homologous recombination repair of double-strand breaks [25] through the activity of a RecA recombinase protein [26] . This same mechanism is exploited during plastome transformation to enable targeted gene replacement between homologous sequences of the artificial DNA and endogenous loci ((1) in Figure 1 ) [27, 28] .

During evolution, plastids largely abandoned the prokaryotic regulation of gene expression via the control of transcription initiation [29] in favor of more elaborated posttranscriptional mechanisms [30] . Through such mechanisms, a tight spatiotemporal coordination of local plastid protein synthesis and import is possible during the biogenesis of photosynthetic complexes [31] . To this end, chloroplasts employ an extended network of nucleus-encoded factors that bind in a highly selective way to the 3 -and 5 -untranslated regions (UTRs) of mRNAs [32] . These RNA-binding proteins are crucial components of the anterograde (nucleus-to-chloroplast) system that regulates the expression of plastid genes, being responsible for stabilizing individual transcripts, orchestrating intron splicing, and promoting the maturation of polycistronic pre-mRNAs [33] . In contrast with plants [34] , RNA editing does not occur in algal chloroplasts [35] . However, it should be possible to engineer this post-transcriptional regulation of transgene expression in microalgal plastids using synthetic RNA-binding proteins [36] . Hence, the chloroplast represents a unique hybrid genetic system with two prominent eukaryotic features: the existence of both group I and group II introns [37] and their complex trans-splicing [38] , and the occurrence of extensive mRNA processing [39] . Finally, the plastid translation machinery is equipped with chaperones [40] , such as disulfide- [41] and peptidylprolyl-isomerases [42] and is thus able to support the native folding of complex recombinant proteins.

Algal plastome transformation was first reported for C. reinhardtii in the late 1980s [43, 44] using the biolistic technique [45] . Alternative protocols include the agitation of cell walldeficient (permeable) genotypes in the presence of glass beads [46, 47] and electroporation [48] . In its classical design, a chloroplast transgenic cassette must include two external homologous flanking regions enclosing a gene of interest (GOI) and a selectable marker (SM), both equipped with a pair of cis-acting regulatory elements: promoter, upstream 5and downstream 3 -UTR ((1) in Figure 1 ). Codon optimization can be easily performed using online tools, such as the Chlamy Sequence Optimizer [49] , while pairs of flanking regions with minimal (≈200 bp) homology [50] are selected ad hoc to introduce transgenes at neutral (non-coding) loci, or to replace disrupted genes. It should be noted, however, that high sequence variability exists between C. reinhardtii ecotypes [10] , thus the same pairs of homology regions may not work with similar efficiency across different strains. Cassettes of higher complexity can be assembled starting from this minimal scaffold to explore combinations of regulatory elements.

Overall, the ability to perform gene targeting, and the absence of epigenetic silencing, render the plastome a "safe harbor" for transgenes and, thus, the expression of recombinant products. This stands in contrast to nuclear engineering, which usually battles with unpredictable transgene integration, resulting in position effects and methylationdependent silencing [51] . Although various approaches were pursued to attenuate these constrains [52] [53] [54] [55] [56] [57] , average cytoplasmic yields are still significantly lower compared to chloroplast expression, where the accumulation of the target product can reach up to 21% of total protein content [58] .

The establishment of transplastomic strains requires, in the first place, reliable selection protocols. The aadA1 (aminoglycoside adenyltransferase, spectinomycin, streptomycin resistance) [59] and aphA6 (aminoglycoside 3 -phosphotransferase, kanamycin resistance) [60] antibiotic detoxifying genes are the typical chloroplast selectable markers. Usually, initial transformants are subcultured for several rounds (6-8) on selective media to enrich the transformed chromosome copies until homoplasmy is reached and experimentally verified by PCR. However, it should be noted that a major pitfall of polyploid genomes is the risk of genetic instability, which can arise from persistent heteroplasmy or from spontaneous interor intrachromosomal rearrangements ((2) in Figure 1 ). Accordingly, strategies should be devised to prevent these stochastic events; for example, by including a constant selective pressure in the cultivation strategy. Clearly, antibiotics are not a suitable option in the management of large-scale cultures. Moreover, antibiotic resistance genes should be abandoned in biotechnology because of health and environmental concerns raised over their potential horizontal transfer to other organisms and diffusion through ecosystems [61] . One solution involves SM removal once homoplasmy is established. This is accomplished by engineering short (400-800 bp), homologous, direct repeats flanking the resistance cassette [62] to promote intramolecular recombination and excision upon removal of the selective pressure. Alternatively, fully "antibiotic-free" selection protocols exploit the complementation of recipient strains affected in essential metabolic activities, usually acetate-requiring strains with lesions in plastid photosynthetic genes. Photosynthesis-competent revertants can be selected following transformation with a functional copy of the missing/disrupted gene and selection of minimal medium. Commonly used strains have deletions in the following loci: atpB (beta subunit of the ATPase complex) [63] , psbA (D1 protein of photosystem II, PSII) [64] , rbcL (RuBisCO large subunit [65] , petB (cytochrome 6b) [66] , tscA (trans-splicing factor) [67, 68] , and psbH (subunit H of PSII) [69] . A recent version of the latter was created by disrupting the psbH locus with the aadA1 cassette in the cell wall-deficient strain cw15 (TN72, CC-5168) [47, 70] . Transformants are obtained via glass agitation by retrofitting the functional psbH gene and, when homoplasmic, are sensitive to spectinomycin due to the loss of the aadA1 cassette. However, despite their widespread use, non-photosynthetic mutants have two main drawbacks: (i) the locus-specific targeting requirement with an ad hoc recipient strain, (ii) the need to include the complementing gene in the transformation cassette.

A recent innovation in algal biotechnology is the adoption of the ptxD gene, encoding the NAD + -dependent phosphite oxidoreductase enzyme from the bacterium Pseudomonas stutzeri [71] . PTXD catalyzes the conversion of phosphite ions (Phi, PO 3 3− ) into phosphate (Pi, PO 4 3− ), making this unharmful, reduced phosphorus compound a very convenient selective agent ((3) in Figure 1 ). In fact, most organisms-including the parasites that commonly infest microalgal cultures-cannot readily assimilate Phi in their metabolism, while PTXD-expressing transgenics can grow when Phi is provided as the sole source of the essential nutrient phosphorus. Accordingly, ptxD was first introduced into the nucleus of C. reinhardtii [72, 73] to develop a Phi-based pest control strategy to enable axenic microalgal cultivation in non-sterile conditions. Chloroplast ptxD expression was subsequently reported in different studies [74] [75] [76] and, recently, an engineered enzyme isoform, more suited for the chloroplast biochemical environment, was developed. This catalytically flexible PTXD version uses NAD + (limiting in the chloroplast) and NADP + (not limiting in the chloroplast) nicotinamide cofactors with equal efficiency and could be reliably employed as a metabolic selectable marker to generate plastid transformants via the direct selection on Phi, both in C. reinhardtii [77] and in the non-model species Picochlorum [78] . In a proof-of-principle approach, PTXD was translationally fused to a GOI, creating an in vivo cleavable protein chimera [79] . In this instance, fertilization with Phi imposes a strict metabolic pressure against the risk of genetic instability, ensuring transgene(s) maintenance in the plastome, and affording the safe upscalability of cultivation systems with a concomitant reduction in management costs.

Efficient heterologous expression requires the association of cis-acting elements to transgenes to regulate their transcription and translation. In addition to promoters, 5 -and 3 -UTRs are critical features of chloroplast engineering [80] , since these sequences are known to play crucial structural roles [81] [82] [83] and influence the stability [84] [85] [86] , lifetime [87, 88] , and translation [89] [90] [91] of endogenous mRNAs. Accordingly, the expression of recombinant products in the chloroplast is significantly influenced by the choice of these regulatory sequences [92] . Traditionally, cis-acting elements derived from photosynthetic genes (atpA, psaA, rbcL, psbD, and psbA), or 16S RNA, are employed to regulate transgene expression [93] . Several efforts were made to define the most efficient regulatory sequences, including the hybrid combinations thereof [90, [94] [95] [96] .

The psbA promoter/5 -UTR pair deserves a special mention. This strong, lightinducible element is known to be repressed by the synthesis of its product through a negative feedback loop acting on the psbA mRNA 5 -UTR [97] . Therefore, when transgenes are placed under the transcriptional control of the psbA promoter, they will likely suffer from this endogenous attenuation, resulting in poor expression. Accordingly, the psbA promoter is commonly used in non-photosynthetic psbA-deficient backgrounds via heterotrophic cultivation, thus restricting the light-driven production of recombinant molecules. A first solution to this issue involved the use the psbD promoter to complement psbA transcription (Manuell et al., 2007) and restore photosynthetic competence while exploiting the psbA element to drive efficient transgene expression. More recently, two studies employed psbA genes from other photosynthetic organisms [98] , including interspecific psbA promoter/5 -UTR combinations [99] , to relieve the interference on transgenes. These experiments clearly showed that it is possible to introduce completely heterologous regulatory elements to drive transgene expression in microalgae. This principle was recently extended to a complete foreign plant system, where nucleus-encoded trans-acting factors from Arabidopsis thaliana and Zea mays were used in C. reinhardtii to stabilize plastid transgenes bearing the native plant recognition sequence on their 5 -UTR [100] .

Altogether, it appears that cis elements can result in high expression variability depending on the associated transgene [86, 95, 101] ; thus, empirical optimization is required. Today, modular cloning techniques enable the seamless fusion of elements, and explore combinatorial cassette design. In this respect, the MoClo (modular cloning) toolkit [102] significantly facilitated nuclear engineering efforts in C. reinhardtii. However, despite earlier attempts [64, 103] , a comprehensive library of validated standard genetic elements and cloning system for algal plastomes is still missing, and is expected to drastically accelerate innovation in this field.

The efforts to improve recombinant protein yields can significantly benefit from reporter systems to non-invasively monitor transgene expression in vivo [9, 104] . The green fluorescent protein (GFP) and its derivatives are particularly useful tools since they enable the rapid screening of transformants via microscopy-or flow cytometry-based techniques. Recent innovations in this respect include chloroplast-derived optimized isoforms of the vivid Verde (VFP, green) [105] and mCherry (red) fluorescent proteins [106] . These reporters can also be employed to assess the impact of different cultivation parameters-mainly light regime, temperature, and metabolism-on recombinant protein expression. For instance, a recent study reported that mixotrophic growth (a combination of heterotrophic acetate fermentative consumption and photoautotrophy) with moderately low light (35 µE m −2 s −1 ) resulted in the highest accumulation of a GFP-fused bacterial lytic protein under the control of the 16S promoter [107] . A novel, high-throughput, fluorescence-based cell sorting system was recently described and validated to select GFP-expressing nuclear transformants of two non-model algal species [108] . This method enables the concomitant assessment of photosynthetic parameters, and is expected to reveal useful applications in transplastomic technology. and serves as a metabolic selectable marker, also enabling axenic algal cultivation in non-sterile media [77] . (4) Transgene expression can be finely controlled via chemical-or temperature-inducible, nucleus-encoded, trans-acting factors that (5) bind the 5 -UTRs of plastid mRNAs and promote their translation [109] . (6) The CITRIC (cold-inducible translational readthrough in chloroplasts) system requires plastome manipulation only and exploits a temperature-sensitive suppressor tRNA to regulate translation [110] . (7) Polycistronic constructs, in which multiple open reading frames (ORFs) are connected via native intercistronic expression elements (IEEs), are processed by endogenous trans-acting factors into separate mono-cistronic transcripts that are independently translated [111] . (8) As pioneered in plant plastids, transgene expression can potentially be achieved in microalgae via episomal vectors that do not require integration into the circular chromosome, but are stably maintained by the host.

The list of recombinant products expressed in the chloroplast of the model species C. reinhardtii embraces several categories, truly testifying the versatility of this heterologous system. A comprehensive list is reported in Table 1 , along with the used selection strategy, cis-regulatory elements, and other relevant information for each work.

A major advantage of microalgae is that the whole harvested biomass contains the product(s) of interest. Following processing, products can be easily retrieved via peptide tags [112, 113] . In this respect, cell wall-deficient mutants are particularly useful, since their cells are easily disrupted with mild forces [114] ; however, this feature also makes them susceptible to the shear forces present during biomass collection [115] . Alternatively, given the non-toxic status [116] and digestibility of most algal species [117] , the biomass can be processed into edible lyophilized capsules that survive the gastric environment, preserving the biological activity of the products [118] .

The possibility to exploit the light-driven conversion of CO 2 into high-value heterologous molecules makes the algal chloroplast an excellent system for the low-cost production of biopharmaceuticals [119, 120] . However, it should be noted that glycosylation, an essential post-translational modification process required for the biological activity of many therapeutical proteins [121] , cannot be achieved in the chloroplast [122] , thus limiting the range of potential products. Nevertheless, successful chloroplast expression was reported for antimicrobial peptides [99, 107, [123] [124] [125] and human proteins, with applications in regenerative medicine [70, [126] [127] [128] and the treatment of hypertension [129, 130] . Notably, algal chloroplasts can assemble full-length human immunoglobulins [131] , including monoclonal antibodies against toxins [132] [133] [134] [135] , whose sequestration in the organelle is beneficial to prevent detrimental effects on eukaryotic cell components.

Arguably, the most promising biomedical application of microalgae is the development of orally deliverable subunit vaccines and other immunizing agents [136] , such as allergens [137] . Examples include epitopes of the foot-and-mouth disease virus [138] , the bacterium Staphylococcus aureus [139] , and diabetes- [140] and atherosclerosis-associated autoantigens [141] . A series of works, instead, produced surface epitopes of the malariacausing agent Plasmodium falciparum [112, 113, [142] [143] [144] [145] [146] that could elicit transmission-blocking antibodies in mice. Importantly, a major constraint of expressing viral antigens in algal chloroplasts is their susceptibility to unspecific proteolytic degradation [58] . This likely explains why the race to develop a vaccine against the SARS-CoV-2 virus has not yet produced a chloroplast-based candidate. Recently, cytoplasmic accumulation of an epitope corresponding to the viral receptor-binding domain (RBD) was achieved following the nuclear transformation of C. reinhardtii. However, when the polypeptide was targeted to the chloroplast, a significant portion of the RBD moiety was lost [147] .

In addition to human health, transplastomic microalgae are finding emerging applications in aquaculture [148] . Few studies reported the production antigens of fish- [95] and shrimp-associated pathogens [58, 149] that efficiently prevent infections. Alongside antigenic peptides, algal chloroplasts are also suitable expression systems from which to manufacture double-stranded RNA targeting common fish viruses [150] . Reported veterinary applications of transplastomic microalgae include the production of swine-associated pathogens [151] and/or enzyme-based dietary supplements for aviculture [152] .

The algal chloroplast has been proposed as a biofactory from which to produce heterologous enzymes with industrial applications [65] , and for biomanufacturing non-native, high-value metabolites. Indeed, the chloroplast harbors an extended metabolic network, into which new enzymes or pathways can be introduced to produce synthetic metabolites. Metabolic engineering in algal plastids is still in its infancy; however, the potential of this organelle to serve as an experimental system for synthetic biology approaches has already been acknowledged [153] . Indeed, the plastid contains several biosynthetic precursors that can be used to produce high-value metabolites. For instance, an early study on C. reinhardtii showed that chloroplast expression of the β-carotene hydroxylase gene (crtR-B) from Haematococcus pluvialis (lacustris) resulted in the overaccumulation of carotenoids, including the potent antioxidant pigment astaxanthin [154] . Recently, the whole astaxanthin pathway operon was introduced into transplastomic tobacco (Nicotiana tabacum) plants, demonstrating the feasibility of complex chloroplast metabolic engineering to produce this heterologous metabolite [155] . Isoprenoids (or terpenes) are another class of metabolites that could be heterologously manufactured in microalgae [156] . These molecules have several applications in human health [157] , and can be produced in plastids, starting from the precursors originating from the endogenous carotenoid biosynthetic pathways. In this respect, two studies have successfully expressed a bifunctional diterpene synthase [158] , and a plant cytochrome P450 [159] , in the chloroplast of C. reinhardtii, showing that these enzymes do not interfere with algal physiology in pilot-scale cultivation [160] .

Another emerging application of transplastomic microalgae is their use in the production of renewable energy through the saccharification of lignocellulose. Initially attempted in the halophytic species Dunaliella tertiolecta [161] , this strategy was subsequently implemented in C. reinhardtii [162, 163] with the expression of hyperthermophilic cellulases [79, 164] . In this application, the dried algal powders containing the recombinant enzymes are mixed at high temperatures with the raw biomass to promote the release of fermentable sugars. To achieve full valorization of lignocellulose, however, additional enzymes with auxiliary activities are required to promote the hydrolysis of recalcitrant components. In this respect, lytic polysaccharide monooxygenases [165] and lignin-modifying enzymes [166] are suitable candidates. These are typically cuproenzymes (copper-requiring), whose production should not be limited by the availability of this element in the chloroplast [167] . Expression of the hydrolytic enzyme is coupled to the PTXD growth selector, enabling selective growth in non-sterile, phosphitefertilized medium [79] P, promoter; UTR, untranslated region.

Full exploitation of transplastomic microalgae requires the ability to introduce multiple open reading frames (ORFs) to implement metabolic pathways and/or enable the synthesis of multi-subunit proteins. To date, microalgal plastome engineering has mostly relied on the insertion of a single transgene coupled to a SM, while multigenic strategies are already established in several plant species [155, [170] [171] [172] .

A relatively simple strategy attempted in microalgae involves the use of endogenous Shine-Dalgarno-like sequences to connect independent ORFs exploiting the prokaryotic mechanism of ribosome reinitiation [124, 129, 141, 173] . Another promising approach is based on polycistronic transgenic units that reflect the endogenous organization and expression of gene batteries in plastids [10, 39] . This strategy was pioneered in plants [171, 174, 175] and was only recently attempted in microalgal plastids [111] . In this system, short intergenic sequences, known as intercistronic expression elements (IEEs), are used to connect multiple ORFs in transgenic operons ((7) in Figure 1 ). IEEs are recognized by nucleus-encoded trans-acting factors that assist the maturation of the polycistronic pre-mRNAs, enabling the independent translation of cistrons [33] . This principle was exploited to express a bicistronic construct composed of a SM and a GFP reporter using two IEEs derived from the endogenous psbN-psbH and tscA-chlN gene pairs [111] .

Alternative strategies are based on the simultaneous delivery of multiple ORFs, either via single [176] or sequential transformation events [177] . It should be noted, however, that the sequential approach is limited by the availability of SMs and cis-regulatory elements. In addition, the repetitive use of genetic elements in the transgenic cassettes may favor spontaneous intramolecular recombination and genetic instability. This issue was highlighted in a recent study [177] , where the association of the rbcL 3 -UTR element in three adjacent ORFs resulted in the loss of two genes, although this unwanted outcome could be prevented by using a shorter version of this sequence.

An earlier synthetic biology approach demonstrated the possibility of performing multigenic engineering using, as a testbed, a series of endogenous photosynthetic genes [176] . Initially, the authors sequentially removed six unrelated genes, and then reintroduced them in the form of single transgenic cluster, where each ORF was equipped with its native cis-regulatory elements; interestingly, the complemented strains displayed suboptimal photosynthetic efficiency compared to the parental genotype. Overall, the task of chloroplast multigenic engineering in C. reinhardtii is open for major improvements.

A desirable feature of heterologous systems is the ability to temporally control transgene expression to avoid potentially toxic effects of foreign peptides (or metabolites) on the host's physiology. In addition, the constitutive hyperaccumulation of recombinant products in the chloroplast can override the quality control machinery and activate the unfolded protein response, leading to their proteolytic degradation [178, 179] .

An early type of inducible system exploited the nucleus-encoded maturation factor Nac2, a trans-acting element belonging to the tetratricopeptide-like protein family that natively stabilizes the 5 -UTR of the plastid psbD transcript [180, 181] to regulate the expression of chloroplast transgenes ((4,5) in Figure 1 ). By placing Nac2 under the transcriptional control of copper- [109, 168, 182] or vitamin B12-repressible promoters [183, 184] , or via fusion to a thiamine pyrophosphate riboswitch [185, 186] , it is possible to tightly regulate the translation of plastid transgenes equipped with the psbD 5 -UTR.

In addition to chemical inducers, temperature-shifts can be used to control transgene expression [187] . Initially, the hybrid Hsp70A-Rbcs2 promoter, which is activated above 40 • C, was used to regulate the expression of the nuclear-encoded protein TDA1 (chloroplast translation factor 1), which in turn stabilizes transgenes bearing its native binding site, the atpA 5 -UTR [188, 189] .

A recently developed temperature-controlled expression system named CITRIC (coldinducible translational readthrough in chloroplasts), instead, requires only plastome engineering [110] ((6) in Figure 1 ). This approach is based on two elements: (i) an amber (UAG) suppressor tRNA gene [190] , which is only stable below 20 • C [191] , and (ii) a GOIharboring multiple UGA within its coding sequence. Notably, none of the plastid genes contains the amber codon [192] , although it is recognized as a terminator. In this system, above 20 • C, the nascent recombinant polypeptide undergoes premature translation abortion, while at lower temperature the engineered tRNA enables its full synthesis. This codon reassignment principle has two additional benefits. On the one hand, it prevents potential toxic effects of leaky chloroplast promoters during cloning operations in bacteria [193] . On the other hand, this strategy can be exploited for biocontainment purposes to reduce the risk of transgene flow and/or escape from genetically engineered microalgae [76, 194] .

A crucial step for the advancement of algal biotechnology is the transition towards non-model, more robust algal species. The phylum Chlorophyta is truly an untapped reservoir of biodiversity, comprising over 4500 species [195] . Many evolved in extreme ecological niches, and possess physiological characteristics that are relevant for biotechnological exploitation. However, because of recalcitrance to transformation and/or a lack of customized tools, only a few microalgal species have been genetically engineered so far [196] . As previously reported, plastid promoters and UTRs from different photosynthetic organisms are interchangeable between species [98] ; however, it is always preferable to use native sequences to drive transgene expression. In this respect, genomic resources are essential tools to enable the customization of cassettes and biotechnological transfer. To this end, the online repository OGDA (organelle genome database for algae, http://ogda.ytu.edu.cn/ accesses on 27 March 2022) [197] enables the visualization of sequenced algal plastomes, while the ChloroMitoCU resource facilitates the selection of synonymous codons for each species [198] .

Of particular interest for biotechnology are microalgae that withstand environmental fluctuations, and with fast generation times. Accordingly, bioprospecting endeavors have focused on resilient species amenable to outdoor cultivation [199] . Reports of chloroplast transformation of non-model species are listed in Table 2 , along with information regarding the insertion loci, regulatory elements, selection strategies, and expressed products. These include halophilic organisms, such as Tetraselmis subcordiformis [200, 201] ; two subspecies of the β-carotene-accumulating Dunaliella genus D. salina [202] and D. tertiolecta [161] ; Desmodesmus armatus [203] ; and the biological producer of the potent antioxidant pigment astaxanthin, H. pluvialis (lacustris) [204] [205] [206] .

Of all Chlorophyta, species belonging to the class Trebouxiophyceae are regarded as the most promising candidates for industrial applications due to their thermotolerance, resistance to extreme irradiance, and high growth rates. For instance, the species Chlorella sorokiniana can be cultivated at very high density under heterotrophic conditions, with reported fresh biomass production of 250 g L −1 in 1000 L fermenters [207] , while its related species C. ohadii thrives at extremely high light intensities due to its exceptionally efficient photoprotective mechanisms [208] . However, despite the suitability of these species for large-scale outdoor cultivation, and the availability of sequenced plastomes [209] , their exploitation in the production of recombinant molecules is currently hampered by recalcitrance to genetic transformation, mostly due the presence of a thick cell wall [210] . So far, successful chloroplast transformation was reported in three Trebouxiophyceae species: C. vulgaris [211] , a species lacking IRs in its plastome [212] ; Parachlorella kessleri [213] ; and the emerging halophilic species, Picochlorum renovo [78, 173] . Chloroplast engineering was also described for stramenopiles, including the oleaginous alga Nannochloropsis oceanica [48] and the closely related species N. gaditiana [214] , and the diatom Phaeodactylum tricornutum [215] . Stramenopiles are currently regarded as promising green biofactories due to their stress-tolerant and high lipid-accumulating phenotype, and their amenability to genetic manipulation due to an expanding genetic engineering toolbox [216] .

Overall, the combined efforts of bioprospecting and genome sequencing, such as in the recent case of the high-light-tolerant chlorophyte, Asterarcys sp. [217] , will favor the domestication [218] of industrially relevant microalgal species, leading to more efficient transplastomic expression platforms. 

Over the last two decades, microalgal research has significantly progressed, and today synthetic biologists have access to a profound knowledge and sophisticated tools to manipulate microalgal plastomes. Since its latest update [219] , the chloroplast engineering toolbox has incorporated novel strategies, enabling the development of robust plastid-based expression platforms with negligible health and environmental risks. A major innovation in this field is the existence of alternatives to antibiotics for creating new strains, such as the PTXD/Phi platform [77] , and the implementation of programmable/inducible systems, such as the temperature-controlled expression of recombinant products [110] .

Undoubtedly, the greatest challenge lying ahead is the ability to perform complex genetic manipulations on algal chloroplasts, including the design of reduced synthetic chromosomes harboring the essential genetic information, along with transgenic elements. An early approach in this direction described the ex vivo assembly in a yeast-bacterial system of a hybrid algal plastome, containing genes from Scenedesmus obliquus, and its insertion into a non-photosynthetic C. reinhardtii strain [220] . Although full complementation could not be obtained in the resulting strain, this work paves the way for whole-plastome engineering in microalgae, and serves as a conceptual basis for further improvements.

Leveraging on recent advancements of plant biotechnology, it should be possible to stably introduce episomal vectors that replicate independently from the native plastome ((8) in Figure 1 ). This strategy was pioneered in tobacco and potato (Solanum tuberosum) plants, with the expression of transgenic cassettes encoded by synthetic mini-chromosomes harboring either a viral [221] or a chloroplast origin of replication [222] . In both cases, episomal expression was stable through generations, as well as after removal of the selective pressure, suggesting that the foreign system was stably maintained by the endogenous plastid DNA replication machinery. This innovative genetic engineering approach is also expected to find immediate applications in microalgae, especially in non-model species that are recalcitrant or not yet amenable to foreign DNA integration in their plastome.

In conclusion, due to these technological perspectives, a bright future awaits microalgal biotechnology, including the commercialization of their derived products. However, the full realization of this goal relies on two important aspects: market acceptance [223] and the enhancement of productivity [224] to withstand competition from traditional heterologous expression systems. 

Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli

Construction of biologically functional bacterial plasmids in vitro

Expression in Escherichia coli of a Chemically Synthesized Gene for the Hormone Somatostatin

Recent advancements in the genetic engineering of microalgae

Gene Delivery Technologies with Applications in Microalgal Genetic Engineering

The unicellular green alga Chlamydomonas reinhardtii as an experimental system to study chloroplast RNA metabolism

Functional Analysis of Plastid Genes through Chloroplast Reverse Genetics in Chlamydomonas

The endosymbiotic origin, diversification and fate of plastids

Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast

High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates

Proteomic Characterization of the Chlamydomonas reinhardtii Chloroplast Ribosome: Identification of proteins unique to the 70 s ribosome

Antibiotic resistance mutations in the chloroplast 16S and 23S rRNA genes of Chlamydomonas reinhardtii: Correlation of genetic and physical maps of the chloroplast genome

Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: Molecular and genetic characterization of integration events

Gene transfer to the nucleus and the evolution of chloroplasts

Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement

The chloroplast proteome: A survey from the Chlamydomonas reinhardtii perspective with a focus on distinctive features

The chloroplast protein import system: From algae to trees

Chloroplast-localized translation for protein targeting in Chlamydomonas reinhardtii

The Chlamydomonas reinhardtii Plastid Chromosome: Islands of Genes in a Sea of Repeats

The Chlamydomonas Chloroplast HLP Protein Is Required for Nucleoid Organization and Genome Maintenance

Chloroplast gene transmission in Chlamydomonas reinhardtii: A random choice model

The Treasure Trove of Algal Chloroplast Genomes. Surprises in Architecture and Gene Content, and Their Functional Implications

Chapter Six-Evolution of the Plastid Genome in Green Algae

Short dispersed repeats in the Chlamydomonas chloroplast genome are collocated with sites for mRNA 3' end formation

Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome

Identification and Expression Analysis of cDNA Encoding a Chloroplast Recombination Protein REC1, the Chloroplast RecA Homologue in Chlamydomonas reinhardtii

Studies on homologous recombination in the green alga Chlamydomonas reinhardtii

Double strand break-induced recombination in Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription

Searching limiting steps in the expression of chloroplast-encoded proteins: Relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii

Chloroplast Biogenesis of Photosystem II Cores Involves a Series of Assembly-Controlled Steps That Regulate Translation

Pas de Trois: An Overview of Penta-, Tetra-, and Octo-Tricopeptide Repeat Proteins From Chlamydomonas reinhardtii and Their Role in

The Expansion and Diversification of Pentatricopeptide Repeat RNA-Editing Factors in Plants

Deep Transcriptome Sequencing of Two Green Algae, Chara vulgaris and Chlamydomonas reinhardtii, Provides No Evidence of Organellar RNA Editing

Small, I. A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

The catalytic group-I introns of the psbA gene of Chlamydomonas reinhardtii: Core structures, ORFs and evolutionary implications

The Chloroplast Trans-Splicing RNA-Protein Supercomplex from the Green Alga Chlamydomonas reinhardtii

Small RNA profiling in Chlamydomonas: Insights into chloroplast RNA metabolism

The Chlamydomonas genome reveals its secrets: Chaperone genes and the potential roles of their gene products in the chloroplast

Protein Disulfide Isomerase as a Regulator of Chloroplast Translational Activation

Chlamydomonas Immunophilins and Parvulins: Survey and Critical Assessment of Gene Models

Chloroplast Transformation in Chlamydomonas with High Velocity Microprojectiles

Studies on Chlamydomonas Chloroplast Transformation: Foreign DNA Can Be Stably Maintained in the Chromosome

Production of Recombinant Proteins in the Chloroplast of the Green Alga Chlamydomonas reinhardtii

Low-Cost Method for Chloroplast Transformation of the Green Alga Chlamydomonas reinhardtii

A Simple Technology for Generating Marker-Free Chloroplast Transformants of the Green Alga Chlamydomonas reinhardtii

Engineering the Chloroplast Genome of Oleaginous Marine Microalga Nannochloropsis oceanica. Front

CSO-A sequence optimization software for engineering chloroplast expression in Chlamydomonas reinhardtii

Minimal Extent of Sequence Homology Required for Homologous Recombination at the psbA Locus in Chlamydomonas reinhardtii Chloroplasts using PCR-generated DNA Fragments

An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas

Generation of Chlamydomonas strains that efficiently express nuclear transgenes

Good News for Nuclear Transgene Expression in Chlamydomonas

The Integration of Multiple Nuclear-Encoded Transgenes in the Green Alga Chlamydomonas reinhardtii Results in Higher Transcription Levels

Cell Synchronization Enhances Nuclear Transformation and Genome Editing via Cas9 Enabling Homologous Recombination in Chlamydomonas reinhardtii

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii

UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems

Factors effecting expression of vaccines in microalgae

Transgenic expression of aminoglycoside adenine transferase in the chloroplast: A selectable marker for site-directed transformation of chlamydomonas

Tools for chloroplast transformation in Chlamydomonas: Expression vectors and a new dominant selectable marker

Protocol measuring horizontal gene transfer from algae to non-photosynthetic organisms

Selectable marker recycling in the chloroplast

Molecular and genetic analysis of the chloroplast ATPase ofChlamydomonas

A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii

Marker-free genetic engineering of the chloroplast in the green microalga Chlamydomonas reinhardtii

The Klebsiella pneumoniae nitrogenase Fe protein gene (nifH) functionally substitutes for the chlL gene in Chlamydomonas reinhardtii

Engineering the chloroplast genome: Techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii

Two adjacent nuclear genes are required for functional complementation of a chloroplast trans-splicing mutant from Chlamydomonas reinhardtii

The 9-kDa phosphoprotein of photosystem: II. Generation and characterisation of Chlamydomonas mutants lacking PSII-H and a site-directed mutant lacking the phosphorylation site

New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii

Purification and Characterization of a Novel Phosphorus-oxidizing Enzyme from Pseudomonas stutzeri WM88

A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae

Phosphite cannot be used as a phosphorus source but is non-toxic for microalgae

Chloroplast engineering of Chlamydomonas reinhardtii to use phosphite as phosphorus source

Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii

Heterologous expression of phosphite dehydrogenase in the chloroplast or nucleus enables phosphite utilization and genetic selection in Picochlorum spp

A chimeric hydrolase-PTXD transgene enables chloroplast-based heterologous protein expression and non-sterile cultivation of Chlamydomonas reinhardtii

Synthetic Oligonucleotide Libraries Reveal Novel Regulatory Elements in Chlamydomonas Chloroplast mRNAs

Functional in vivo analyses of the 3' flanking sequences of the Chlamydomonas chloroplast rbcL and psaB genes

Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon

The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii

The sequence and structure of the 3 -untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability

Specific roles of 5 RNA secondary structures in stabilizing transcripts in chloroplasts

Redundant cis-acting determinants of 3 processing and RNA stability in the chloroplast rbcL mRNA of Chlamydomonas

Gene elements that affect the longevity of rbcL sequence-containing transcripts in Chlamydomonas reinhardtii chloroplasts

Changes in the 5'-untranslated region of the rbcL gene accelerate transcript degradation more than 50-fold in the chloroplast of Chlamydomonas reinhardtii

Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5' untranslated region

Contribution of 5 -and 3 -untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes

Efficient translation in chloroplasts requires element(s) upstream of the putative ribosome binding site from atpI

Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast

Transcriptional Analysis of Endogenous and Foreign Genes in Chloroplast Transformants of Chlamydomonas

Expression of a foreign gene in Chlamydomonas reinhardtii chloroplast

Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas

Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5 untranslated region optimization

Processing of the psbA 5 Untranslated Region in Chlamydomonas reinhardtii Depends upon Factors Mediating Ribosome Association

Analysis of heterologous regulatory and coding regions in algal chloroplasts

Production of recombinant proteins in microalgae at pilot greenhouse scale

Use of plant chloroplast RNA-binding proteins as orthogonal activators of chloroplast transgenes in the green alga Chlamydomonas reinhardtii

Effect of coding regions on chloroplast gene expression in Chlamydomonas reinhardtii

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii

Gateway-Assisted Vector Construction to Facilitate Expression of Foreign Proteins in the Chloroplast of Single Celled Algae

Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast

Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter

mCherry Protein as an In Vivo Quantitative Reporter of Gene Expression in the Chloroplast of Chlamydomonas reinhardtii

Optimising light conditions increases recombinant protein production in Chlamydomonas reinhardtii chloroplasts

Droplet-based microfluidic screening and sorting of microalgal populations for strain engineering applications

Regulated Chloroplast Gene Expression in Chlamydomonas

Cold-inducible translational readthrough in the chloroplast of Chlamydomonas reinhardtii using a novel temperature-sensitive transfer

Intercistronic expression elements (IEE) from the chloroplast of Chlamydomonas reinhardtii can be used for the expression of foreign genes in synthetic operons

Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii

Light-Induced Production of An Antibody Fragment and Malaria Vaccine Antigen from Chlamydomonas reinhardtii

Mild and Selective Protein Release of Cell Wall Deficient Microalgae with Pulsed Electric Field

Downstream Processing of Chlamydomonas reinhardtii TN72 for Recombinant Protein Recovery

A Toxicological Evaluation of Chlamydomonas reinhardtii, a Green Algae

Microalgae of interest as food source: Biochemical composition and digestibility

An evaluation of microalgae as a recombinant protein oral delivery platform for fish using green fluorescent protein (GFP)

The algal chloroplast as a platform for making biopharmaceuticals

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

Appropriate Glycosylation of Recombinant Proteins for Human Use: Implications of Choice of Expression System

Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals. Front

Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

Production of milk-derived bioactive peptides as precursor chimeric proteins in chloroplasts of Chlamydomonas reinhardtii

Synthesis of bacteriophage lytic proteins against Streptococcus pneumoniae in the chloroplast of Chlamydomonas reinhardtii

Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii

Bioactivity of an antihypertensive peptide expressed in Chlamydomonas reinhardtii

Expression of multiple antihypertensive peptides as a fusion protein in the chloroplast of Chlamydomonas reinhardtii

Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplasts

Production of anti-cancer immunotoxins in algae: Ribosome inactivating proteins as fusion partners

Production of unique immunotoxin cancer therapeutics in algal chloroplasts

Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin

Efficient targeting of recombinant proteins to the thylakoid lumen in Chlamydomonas reinhardtii using a bacterial Tat signal peptide

Algae-based oral recombinant vaccines

Expression and Characterization of Functional Recombinant Bet v 1.0101 in the Chloroplast of Chlamydomonas reinhardtii

Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast

Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65)

Chlamydomonas reinhardtii chloroplasts express an orally immunogenic protein targeting the p210 epitope implicated in atherosclerosis immunotherapies

Algae-Produced Pfs25 Elicits Antibodies That Inhibit Malaria Transmission

Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines

Alga-Produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated with a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium falciparum Infection of Mosquitoes

Engineering the chloroplast of Chlamydomonas reinhardtii to express the recombinant PfCelTOS-Il2 antigen-adjuvant fusion protein

Expression of Recombinant PfCelTOS Antigen in the Chloroplast of Chlamydomonas reinhardtii and its Potential Use in Detection of Malaria

Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

An oral delivery system for controlling white spot syndrome virus infection in shrimp using transgenic microalgae

Enhanced production of antiviral dsRNA in the Chlamydomonas reinhardtii chloroplast via a novel convergent promoter expression system

Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas reinhardtii chroloplasts

Transgenic microalgae expressing Escherichia coli AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks

The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism

Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii

Riboswitch-mediated inducible expression of an astaxanthin biosynthetic operon in plastids

Eukaryotic microalgae as hosts for light-driven heterologous isoprenoid production

Therapeutic and Medicinal Uses of Terpenes

Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii

Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii

Pilot-scale cultivation of wall-deficient transgenic Chlamydomonas reinhardtii strains expressing recombinant proteins in the chloroplast

Production of recombinant enzymes in the marine alga Dunaliella tertiolecta

Comparison of transplastomic Chlamydomonas reinhardtii and Nicotiana tabacum expression system for the production of a bacterial endoglucanase

A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts

A microalgal-based preparation with synergistic cellulolytic and detoxifying action towards chemical-treated lignocellulose

Lytic polysaccharide monooxygenases and other histidinebrace copper proteins: Structure, oxygen activation and biotechnological applications

Ligninolytic enzymes and its mechanisms for degradation of lignocellulosic waste in environment. Heliyon 2020, 6, e03170

From economy to luxury: Copper homeostasis in Chlamydomonas and other algae

Toward mosquito control with a green alga: Expression of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas

Expression of a Synthetic Gene for the Major Cytotoxin (Cyt1Aa) of Bacillus thuringiensis subsp. israelensis in the Chloroplast of Wild-Type Chlamydomonas

A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop

Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons

Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids

Development of a high-productivity, halophilic, thermotolerant microalga Picochlorum renovo

Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons

Stabilization and translation of synthetic operon-derived mRNA s in chloroplasts by sequences representing PPR protein-binding sites

Refactoring the Six-Gene Photosystem II Core in the Chloroplast of the Green Algae Chlamydomonas reinhardtii

Multigenic engineering of the chloroplast genome in the green alga Chlamydomonas reinhardtii

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control

The Mars1 kinase confers photoprotection through signaling in the chloroplast unfolded protein response

Mutation at the chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II

The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability

Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas

Algae acquire vitamin B12 through a symbiotic relationship with bacteria

Unraveling Vitamin B12-Responsive Gene Regulation in Algae

Controlling Expression of Genes in the Unicellular Alga Chlamydomonas reinhardtii with a Vitamin-Repressible Riboswitch

Thiamine biosynthesis in algae is regulated by riboswitches

Light and heat-shock mediated TDA1 overexpression as a tool for controlled high-yield recombinant protein production in Chlamydomonas reinhardtii chloroplasts

Evidence for regulatory function of nucleus-encoded factors on mRNA stabilization and translation in the chloroplast

Dual functions of the nucleus-encoded factor TDA1 in trapping and translation activation of atpA transcripts in Chlamydomonas reinhardtii chloroplasts

Chloroplast heteroplasmicity is stabilized by an amber-suppressor tryptophan tRNA(CUA)

Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae

Function of 3' non-coding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii

Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii

Environmental risks of large scale cultivation of microalgae: Mitigation of spills

How many species of algae are there?

Methodological review of genetic engineering approaches for non-model algae

OGDA: A comprehensive organelle genome database for algae

Codon patterns across organelle genomes for functional genomics and evolutionary applications

Strategies for Bioprospecting Microalgae for Potential Commercial Applications

Chloroplast Transformation of Platymonas (Tetraselmis) subcordiformis with the bar Gene as Selectable Marker

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators

Construction of rice site-specific chloroplast transformation vector and transient expression of EGFP gene in Dunaliella salina

Nuclear and chloroplast genome engineering of a productive non-model alga Desmodesmus armatus: Insights into unusual and selective acquisition mechanisms for foreign DNA

Chloroplast genetic tool for the green microalgae haematococcus pluvialis (chlorophyceae, volvocales)(1)

Over-accumulation of astaxanthin in Haematococcus pluvialis through chloroplast genetic engineering

Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green alga Chlorella sorokiniana for biomass production

The desert green algae Chlorella ohadii thrives at excessively high light intensities by exceptionally enhancing the mechanisms that protect photosynthesis from photoinhibition

The complete chloroplast genome sequence of Chlorella vulgaris and phylogenetic analysis

Insights into cell wall disintegration of Chlorella vulgaris

The chloroplast genetic engineering of a unicellular green alga Chlorella vulgaris with two foreign peptides co-expression

Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris: The existence of genes possibly involved in chloroplast division

Characterization of the Chloroplast Genome Facilitated the Transformation of Parachlorella kessleri-I, A Potential Marine Alga for Biofuel Production

Plastid Engineering of a Marine Alga, Nannochloropsis gaditana, for Co-Expression of Two Recombinant Peptides

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum

Stramenopile microalgae as "green biofactories" for recombinant protein production

The chloroplast genome of a resilient chlorophycean microalga Asterarcys sp

Biomass from microalgae: The potential of domestication towards sustainable biofactories. Microb

Selectable Markers and Reporter Genes for Engineering the Chloroplast of Chlamydomonas reinhardtii

An exogenous chloroplast genome for complex sequence manipulation in algae

Replicating minichromosomes as a new tool for plastid genome engineering

Mini-synplastomes for plastid genetic engineering

Improving Microalgae Research and Marketing in the European Atlantic Area: Analysis of Major Gaps and Barriers Limiting Sector Development

Synthetic Biology Approaches To Enhance Microalgal Productivity

Informed Consent Statement: Not applicable.

The authors declare no conflict of interest.